muramidase and hexylene-glycol

The influence of different parameters on the silicification procedure using lysozyme is reported. When polyethoxysiloxane (PEOS), an internally crosslinked silica reservoir, is used, regular structures with a narrow size distribution could be obtained only via introducing the silica precursor in two steps including initial dropping and subsequent addition of residual oil phase in one portion. We found that mixing sequence of mineralizing agents in the presence of a positively charged surfactant plays a key role in terms of silica precipitation when tetraethoxyorthosilicate (TEOS) is the oil phase. In contrast, well-mineralized crumpled features with high specific surface area could be synthesized in the presence of PEOS as a silica precursor polymer, regardless of mixing sequence. Moreover, introducing sodium dodecyl sulfate (SDS) as a negatively charged surfactant resulted in regular silica sphere formation only in combination with hexylene glycol (MPD) as a specific co-solvent. Finally, it is demonstrated that by inclusion of different nanoparticles even more sophisticated hybrid materials can be generated.

Topics: Adsorption; Animals; Cetrimonium; Cetrimonium Compounds; Chemistry Techniques, Synthetic; Chickens; Glycols; Muramidase; Nanoparticles; Particle Size; Silicon Dioxide; Siloxanes; Sodium Dodecyl Sulfate; Surface Properties; Surface-Active Agents

Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2,4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture. Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with (R)-MPD and (RS)-MPD the crystal contacts are made by (R)-MPD, demonstrating that there is preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization.

Topics: Crystallography, X-Ray; Glycols; Muramidase; Protein Structure, Tertiary

Currently, the investigation of protein refolding processes involves several time-consuming stages that require large amounts of protein and costly chemicals. Consequently, there is great interest in developing new approaches to the study of protein renaturation that are more technically and economically feasible. It has recently been reported that certain cosolvents are able to modulate the denaturing properties of sodium dodecyl sulfate (SDS) and induce the refolding of proteins. This unit presents a protocol to study and follow the renaturation of a protein (membrane or soluble) starting from a native or SDS-unfolded state using a variety of candidate cosolvents and osmolytes.

Topics: Detergents; Enzyme Assays; Glycols; Muramidase; Osmolar Concentration; Protein Denaturation; Protein Renaturation; Proteins; Sodium Dodecyl Sulfate; Solutions; Solvents

Focused acoustic energy allows accurate and precise liquid transfer on scales from picolitre to microlitre volumes. This technology was applied in protein crystallization, successfully transferring a diverse set of proteins as well as hundreds of precipitant solutions from custom and commercial crystallization screens and achieving crystallization in drop volumes as small as 20 nl. Only higher concentrations (>50%) of 2-methyl-2,4-pentanediol (MPD) appeared to be systematically problematic in delivery. The acoustic technology was implemented in a workflow, successfully reproducing active crystallization systems and leading to the discovery of crystallization conditions for previously uncharacterized proteins. The technology offers compelling advantages in low-nanolitre crystallization trials by providing significant reagent savings and presenting seamless scalability for those crystals that require larger volume optimization experiments using the same vapor-diffusion format.

Topics: Acoustics; Animals; Chickens; Crystallization; Crystallography, X-Ray; Egg White; Glycols; Hepacivirus; HIV Reverse Transcriptase; Humans; Muramidase; Nanoparticles; Nanotechnology; Protein-Tyrosine Kinases; Proteins; Serum Albumin; Viral Proteins; Viscosity

Recently, we reported a unique approach to preserve the activity of some proteins in the presence of the denaturing agent, Sodium Dodecyl Sulfate (SDS). This was made possible by addition of the amphipathic solvent 2,4-Methyl-2-PentaneDiol (MPD), used as protecting but also as refolding agent for these proteins. Although the persistence of the protein activity in the SDS/MPD mixture was clearly established, preservation of their structure was only speculative until now.. In this paper, a detailed X-ray study addresses the pending question. Crystals of hen egg-white lysozyme were grown for the first time in the presence of MPD and denaturing concentrations of SDS. Depending on crystallization conditions, tetragonal crystals in complex with either SDS or MPD were collected. The conformation of both structures was very similar to the native lysozyme and the obtained complexes of SDS-lysozyme and MPD-lysozyme give some insights in the interplay of protein-SDS and protein-MPD interactions.. This study clearly established the preservation of the enzyme structure in a SDS/MPD mixture. It is hypothesized that high concentrations of MPD would change the properties of SDS and lower or avoid interactions between the denaturant and the protein. These structural data therefore support the hypothesis that MPD avoids disruption of the enzyme structure by SDS and can protect proteins from SDS denaturation.

Topics: Animals; Chickens; Crystallization; Crystallography, X-Ray; Glycols; Muramidase; Protein Conformation; Protein Denaturation; Sodium Dodecyl Sulfate; Solvents; Surface-Active Agents

Sodium dodecyl sulfate (SDS) is a highly effective and widely used protein denaturant. We show that certain amphipathic cosolvents such as 2-methyl-2,4-pentanediol (MPD) can protect proteins from SDS denaturation, and in several cases can refold proteins from the SDS-denatured state. This cosolvent effect is observed with integral membrane proteins and soluble proteins from either the alpha-helical or the beta-sheet structural classes. The SDS/MPD system can be used to study processes involving native protein states, and we demonstrate the reversible thermal denaturation of the outer membrane protein PagP in an SDS/MPD buffer. MPD and related cosolvents can modulate the denaturing properties of SDS, and we describe a simple and effective method to recover refolded, active protein from the SDS-denatured state.

Topics: Acyltransferases; Animals; Bacterial Outer Membrane Proteins; Bacteriorhodopsins; Buffers; Carbonic Anhydrase II; Chickens; Dose-Response Relationship, Drug; Escherichia coli Proteins; Glycols; Halobacterium salinarum; Hot Temperature; Humans; Hydrophobic and Hydrophilic Interactions; Inclusion Bodies; Muramidase; Protein Conformation; Protein Denaturation; Protein Folding; Protein Renaturation; Protein Structure, Secondary; Sodium Dodecyl Sulfate; Solubility; Solvents; Surface-Active Agents

Hen egg-white lysozyme has been crystallized at slightly alkaline pH using 2-methyl-2,4-pentanediol (MPD) as the precipitant. The crystals are nearly isomorphous to crystals grown at acidic pH using sodium chloride as the precipitant. However, the growth kinetics differ markedly between the two conditions. The major reason for this is a molecule of MPD that binds tightly in between two lysozyme molecules and favors the growth of the crystals along the crystallographic c direction over growth perpendicular to it.

Topics: Animals; Chemical Precipitation; Chickens; Crystallization; Crystallography, X-Ray; Female; Glycols; Hydrogen-Ion Concentration; Muramidase; Protein Conformation; Static Electricity